Gene expression is precisely controlled at all levels and modulation of mRNA stability is an important regulatory step that dictates the ultimate accumulation and functionality of mRNA. Indeed, mRNA molecules have distinct intrinsic half-lives that can span from minutes to several days and can vary in response to different physiological states. The goal of this RFA (DA-18-009) is to identify RNA modifications involved in HIV function to lay the foundation for development of novel therapeutics to treat people dealing with HIV infection and substances of abuse. Here we focus on evaluating a newly identified 5' cap, Nicotinamide Adenine Dinucleotide (NAD+), for its ability to influence both HIV and host RNA expression in the background of HIV, antiretroviral drugs (ART) and cigarette smoke condensate (CSC). The first 5' mRNA cap to be identified was N7- methyl guanosine (m7G) which among other functions imparts stability onto mRNA molecules. Additional caps have subsequently been reported with the most recent identified by Dr. Kiledjian and colleagues as a 5' end NAD+ cap in eukaryotes that can promote RNA decay. Building on this work, the first goal of this proposal is to evaluate the NAD+ 5' cap mRNA modification as it impacts the decay of both host and HIV-specific mRNAs during an HIV infection of CD4+ T cells. In addition, it is important to consider that inflammation may influence RNA modifications, and drugs of abuse in HIV-infected patients are particularly problematic in this regard. This proposal will focus on how cigarette smoke can influence the NAD+ 5' cap RNA modification. Smoking is particularly relevant as an estimated 40-70% of HIV+ patients smoke, representing a prevalence rate 2-3-fold higher than in the general population. Therefore, the second goal of this proposal is to evaluate the impact of CSC on the ability of both host and HIV-specific mRNAs to be NAD+ 5' cap modified. The two aims of the proposal are: 1. Evaluate the ability of ART treatment and CSC to influence NAD+-5' capping of HIV-specific RNA (Aim 1), and 2. Quantify NAD+-5' capping of cellular RNA following HIV infection, ART treatment and in presence of cigarette smoke condensate (Aim 2). This proposal presents an experimental plan that will utilize the expertise that exists between two laboratories to address the experimental objectives; Dr. Sodora's laboratory (HIV/SIV) and Dr. Kiledjian's laboratory (mRNA stability and decay). This R21 will undertake a series of vitro/ex vivo experiments to provide insights into the modifications to host and HIV- specific mRNAs in presence of both cART and cigarette smoke. Our long-term goal is to identify if there is a role for the NAD+ 5'cap on HIV specific and/or cellular RNAs following both HIV infection and exposure to cigarette smoke. These studies have the potential to identify the role of this novel 5' cap to impact RNA decay and to identify a mechanism to explain HIV latency and/or how the HIV virus alters cellular RNA to maximize viral production.